System and method for processing bone marrow

ABSTRACT

A system for collecting and processing donated bone marrow comprises a closed and/or sealable collection container and a closed and/or sealable satellite container. The system may also include at least one porous medium interposed between the collection container and the satellite container. The porous medium is a size gradient depletion media. The system may also include at least one third container in fluid communication with the collection container. The system may also include at least one manifold interposed in the fluid flow path between the collection container and the third container.

TECHNICAL FIELD

This invention relates to a system for processing bone marrow donatedfor the purpose of therapeutic transfusion, for separating the bonemarrow composition into one or more constituents, and, particularly, toimproved methods and apparatuses for preparing and processing a bonemarrow composition. This invention also relates to a biological fluidprocessing system for processing biological fluid into its variouscomponents.

BACKGROUND OF THE INVENTION

Bone marrow is a spongy tissue found inside bones. The bone marrow inthe breast bone, skull, hips, ribs, and spine contains stem cells. Stemcells produce the body's blood cells, e.g., leukocytes, that fightinfection); erythrocytes, that carry oxygen to and remove waste productsfrom organs and tissues; and platelets, that enable the blood to clot.

Bone marrow, and some of the cells and cell types that are present inbone marrow, have been found to have significant therapeutic efficacy inthe treatment of a number of diseases and conditions. For example, stemcells may be separated from the bone marrow composition and may be usedin the treatment of many diseases. Filtered marrow itself may be used asa transplant graft in the treatment of leukemia, aplastic anemia,various lymphomas (e.g., Hodgkin's disease), multiple myeloma, certainimmunodeficiencies, and a variety of cancers (typically solid tumors,such as breast and ovarian cancer).

The development of plastic blood collection bags has facilitated theseparation of donated bone marrow into its various components andanalogous products, thereby making these different components availableas a transfusion product.

Further, significant improvements in reducing the immunogenicity of bonemarrow transplanted into a patient has greatly increased the use of bonemarrow as a competent and desirable therapeutic composition.

For these and other reasons, harvesting bone marrow and separating thebone marrow into components has substantial therapeutic and monetaryvalue. This is nowhere more evident than in treating the increaseddamage to a patient's immune system caused by the higher doses andstronger drugs now used during chemotherapy for cancer patients. Thesemore aggressive chemotherapy protocols are directly implicated in thereduction of the platelet content of the blood to abnormally low levels.

A conventional bone marrow collection procedure may include thefollowing:

(1) Under general anesthesia, a bone marrow aspiration needle isinserted into the iliac crest (the cavity of the rear hip bone) where alarge quantity of bone marrow is located. The bone marrow is extractedwith a needle and syringe. Several skin punctures on each hip andmultiple bone punctures are usually required to extract the requisiteamount of bone marrow.

(2) About 10–30 cc of bone marrow are drawn from each bone puncture, andalthough the total amount drawn is variable (depending primarily on thesize of the donor and the concentration of the bone marrow cells),usually about 1 liter and up to 1.5 liters of bone marrow are harvested.

(3) Each syringe of bone marrow drawn from the donor is thenindividually expressed into an open collection bag or an open container.The collection bag typically includes an anti-coagulant solution such asheparin and/or CPDA-1.

(4) The used syringe is then used to draw anti-coagulant solution intothe syringe, the solution is expelled, and the syringe is used again todraw bone marrow. The process is repeated 30–50 times or more until upto about 1.5 liters of bone marrow is harvested.

(5) During or near the end of the collection process, a sample of about5 cc is then drawn from the collection bag or container, and the amountof stem cells in the sample is either determined or inferred. Harvestingis usually completed when the sample contains about 1–3×10⁸ nucleatedcells per kilogram of body weight of the recipient.

(6) The harvested bone marrow composition is then filtered, generallywithin about 6 to 8 hours of harvesting. The filtration process usuallyinvolves a series of filters having different pore size ratings,typically 850μ, 500μ, and 200μ. These filters remove bone fragments,microaggregates, blood clots, and other undesirable debris from the bonemarrow composition.

(7) The filtered bone marrow is then processed according to its end use.For example, if the bone marrow will be used in an autologoustransplant, the filtered bone marrow will be frozen (cryopreserved) andstored at a temperature between about −80° C. and about −196° C. untilthe day of the transplant. For allogeneic transplants, the bone marrowwill be treated to remove T-cells, then transferred directly to aninfuser for administration to the patient.

The existing regimen for harvesting bone marrow is time-consuming andcostly. Further, the present state of the art involves the use of opencontainers and/or open systems.

In view of this, there is a growing need for an efficient system andmethod for collecting and processing bone marrow, and for harvestingbone marrow in a closed or sealed system.

The devices and methods of this invention alleviate the above-describedproblems and, in addition, provide a higher yield of superior qualitybone marrow.

A problem attendant with the separation of various blood and bone marrowcomponents using a multiple bag system is that the highly valuablecomponents become trapped in the conduits connecting the various bagsand in the various devices that may be used in the system. Conventionalprocessing and storage techniques contribute to these problems. Forexample, air, in particular oxygen, present in stored blood and bloodcomponents, or in the storage container, may lead to an impairment ofthe quality of the blood components, and may decrease their storagelife. More particularly, oxygen may be associated with an increasedmetabolic rate (during glycolysis), which may lead to decreased storagelife, and decreased viability and function of whole blood cells. Forexample, during storage red blood cells metabolize glucose, producinglactic and pyruvic acids. These acids decrease the pH of the medium,which in turn decreases metabolic functions. Furthermore, the presenceof air or gas in the satellite bag may present a risk when a patient istransfused. For example, as little as 5 ml of air or gas may causesevere injury or death. Despite the deleterious effect of oxygen onstorage life and the quality of bone marrow blood and bone marrowcomponents, the prior art has not addressed the need to remove gasesfrom bone marrow processing systems during collection and processing.

Because of the high cost and limited availability of bone marrowcomponents, a device comprising a porous medium used to depleteleucocytes from biological fluid should deliver the highest possibleproportion of the component present in the donated bone marrow. An idealdevice for the filtering or leucocyte depletion of bone marrow would beinexpensive, relatively small, and be capable of rapidly processing thecomponents obtained from about one unit or more of biological fluid(e.g., donated bone marrow), in, for example, less than about one hour.Ideally, this device would reduce the leucocyte content to the lowestpossible level, while maximizing the yield of a valuable blood componentwhile minimizing an expensive, sophisticated, labor intensive effort bythe operator of the system. The yield of the bone marrow or a componentshould be maximized while at the same time delivering a viable andphysiologically active component. It may also be preferable that thebone marrow filter or porous medium be capable of removing platelets, aswell as fibrinogen, fibrin strands, tiny fat globules, and othercomponents such as microaggregates which may be present in the bonemarrow.

Definitions

The following definitions are used in reference to the invention:

(A) Bone marrow or Biological Fluid is the soft tissue within bonecavities, and typically contains whole blood, hematopoietic precursorcells and hematopoietic cells that are maturing into erythrocytes, fivetypes of leukocytes, and thrombocytes. Harvested bone marrow typicallyincludes these components, as well as bone chips, megakaryocytes, stemcells, fat globules, blood clots, fibrin, platelets, among otherbiological and/or cellular matter. Bone marrow or biological fluid alsoincludes any treated or untreated fluid associated with livingorganisms, particularly bone marrow, including harvested unseparated(whole) bone marrow, warm or cold bone marrow, cryopreserved bonemarrow, and stored or fresh bone marrow, treated bone marrow, such asbone marrow diluted with a physiological solution, including but notlimited to saline, nutrient, and/or anticoagulant solutions; one or morebone marrow components, such as stem cells; and analogous bone marrowproducts derived from bone marrow or a bone marrow component. Thebiological fluid may include leucocytes, or may be treated to removeleucocytes. As used herein, bone marrow product or biological fluidrefers to the components described above, and to similar bone marrowproducts or biological fluids obtained by other means and with similarproperties. In accordance with the invention, each of these bone marrowproducts or biological fluids is processed in the manner describedherein.

A typical harvesting procedure commonly draws up to about 1.5 liters ofa composition containing bone marrow from the donor into a bag whichcontains an anticoagulant to prevent the bone marrow from clotting.However, the amount drawn differs from patient to patient and donationto donation.

(B) Filtered bone marrow: refers to a composition containing bone marrowand stem cells that is suitable for use as a transplant graft or anorgan graft. The bone marrow harvested from a donor is treated andprocessed using various regimens before it is suitable for use as atransplant graft. For example, the harvested bone marrow may be mixedwith a nutrient and/or anti-coagulant solution, and may be filtered toremove debris and the like.

(C) Porous medium: refers to the porous medium through which one or morebone marrow, bone marrow components, or biological fluids pass. Atypical porous medium is a filter for removing undesirable constituents,such as pieces of bone, microaggregates, blood clots, and the like, fromthe bone marrow. The bone marrow processing system may optionallyinclude a leucocyte depletion filter or porous medium, which refersgenerically to any one of the media which deplete leucocytes from thebone marrow or a bone marrow component.

The porous media according to the invention may be connected to aconduit interposed between the containers, and may be positioned in ahousing which in turn can be connected to the conduit. As used herein,filter assembly refers to the porous medium positioned in a suitablehousing. An exemplary filter assembly may include pore size filterassembly and/or a leucocyte depletion assembly or device. A biologicalfluid processing system, such as a bone marrow collection and processingsystem, may comprise porous media, preferably as filter assemblies.

SUMMARY OF THE INVENTION

In the devices and methods of the present invention, bone marrow isharvested from a donor and collected or pooled in a collection bag.Typically, the bone marrow is filtered at the time of processing, which,in the United States, is generally within about 6 to 8 hours of the timethe bone marrow is harvested. Thus, as a biological fluid is transferredfrom the collection bag, debris and other undesirable constituents maybe removed by the appropriate porous medium, and filtered bone marrow iscollected in the satellite bag.

In accordance with the invention, a system is provided whereby abiological fluid such as bone marrow or a bone marrow composition isprocessed to form a bone marrow composition suitable for transplantationinto a recipient, or is processed into one or more of its components. Anapparatus and system according to the invention includes a closedcollection container having at least one access port and a firstconnector for communicating with a second, transfer, or satellitecontainer; and a transfer or satellite bag configured to establish fluidflow between the first container and the second container.

Some embodiments of the invention also include at least one filterassembly interposed in the fluid flow path between the first containerand the second container. The filter assembly may be configured toestablish and receive fluid flow from the collection container, saidfilter assembly having at least one porous medium through which bonemarrow passes, said filter assembly may also be configured to establishfluid flow into a transfer or satellite bag.

Some embodiments of the invention also include at least one thirdcontainer, preferably in fluid communication with the first container.Typically, the third container will include an anti-coagulant solution,a nutrient solution, a rinsing solution, a priming solution, or thelike.

Some embodiments of the invention also include at least one manifoldinterposed between the first container and the second container. Themanifold may be configured to establish a fluid flow path into the firstcontainer, into the third container, and/or between the first containerand the second container.

for transferring the harvested bone marrow from the syringe to thecollection bag. In preferred embodiments of the invention, the manifoldincludes one or more ports for transferring bone marrow, harvested fromthe donor in a harvesting container (typically a syringe), into thefirst or collection container.

Additionally, processes and systems according to the invention mayinclude a gas outlet that allows gas that may be present in the systemout of the system or a component of the system.

Processes and systems according to the invention may also include a gasinlet that allows gas into the system to recover a biological fluid thatmay be entrapped or retained during processing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an embodiment of a biological fluid processing systemaccording to the invention.

FIG. 2 is an embodiment of a manifold for transferring bone marrow intoa processing system according to the invention.

SPECIFIC DESCRIPTION OF THE INVENTION

The present invention involves a bone marrow collection and processingassembly or system comprising a closed or sealed first container and aclosed or sealed second container, and a conduit interconnecting thefirst container with the second container, said first container havingat least one access port.

Some embodiments of the invention also include at least one first porousmedium interposed in the fluid flow path between the first container andthe second container. The porous medium, preferably housed in a chamberto form a filter assembly, may include a microaggregate filter elementand/or a gel pre-filter element. In a preferred embodiment of theinvention, the system includes at least one microaggregate filter havinga pre-determined pore size; in a most preferred embodiment of theinvention, the system includes at least one microaggregate filter havinga pore size selected from 850μ, 500μ, and 200μ, or combinations thereof.

Some embodiments of the invention also include at least one thirdcontainer in fluid communication with the first container. Typically,the third container will include an anti-coagulant solution, a nutrientsolution, a rinsing solution, a priming solution, or the like. It isintended that the invention should not be limited by the type or use ofsolution in the third container.

Some embodiments of the invention also include at least one manifoldinterposed in the fluid flow path between the first container and thethird container. The manifold may be variously configured andmulti-functional. For example, the manifold may include at least oneport adapted to engage a portion of the harvesting container, said portbeing configured to establish a fluid flow path between the harvestingcontainer and the first container. The port may also be configured toestablish a fluid flow path between the harvesting container and thethird container. For example, it may be desirable to transfer a bonemarrow composition in a syringe (e.g., a harvesting container) to thefirst container or a collection container, and for drawing andexpressing anti-coagulant solution into and out of the syringe.

In the embodiments of the invention that include a manifold, themanifold may be variously configured. In accordance with the presentinvention, the manifold may be configured or adapted to provide one ormore of the following: a fluid flow path between the harvestingcontainer and the collection container, a fluid flow path between theharvesting container and a third container, a fluid flow path betweenthe third container and the collection container, or combinationthereof. In a preferred embodiment of the invention, the manifold isconfigured to provide a individual fluid flow paths between theharvesting container, the collection container, and the third container.

A system according to the invention also includes one or more flowrestrictors so that the collection container, the filter assembly, thetransfer container, the third container, the manifold, or combinationsthereof may be separated from the system.

The bone marrow collection and processing system may also include one ormore fluid flow valves for opening and/or closing a fluid flow pathanywhere in the system, or for channeling fluid through a specific fluidflow path. Exemplary fluid flow valves include but are not limited to astopcock, a plunge valve, and a re-sealable septum valve. The fluid flowvalves may by unidirectional or multi-directional. In some of the fluidflow valves, it may be desirable that the valve is re-sealable orclosable.

As shown in more detail below, the assembly may also include additionalcontainers, porous media, and conduits interconnecting the variouselements of the system or assembly.

The invention also involves a method for collecting and processing bonemarrow comprising harvesting bone marrow from a donor, collecting thebone marrow in a first closed or sealed container; passing the bonemarrow through at least one porous medium, the porous medium comprisinga microaggregate filter having a pre-determined pore size or rating; andpassing the filtered bone marrow into a second closed or sealedcontainer. In a preferred embodiment of the invention, the porous mediumis positioned in a housing to form a filter assembly, and the filterassembly is interposed in the fluid flow path between the firstcontainer and the second container. In a most preferred embodiment ofthe invention, multiple filter assemblies are interposed in the fluidflow path between the first container and the second container, eachfilter assembly having a porous medium of different pore rating than theother porous media. In general, harvested bone marrow is processed orfiltered as soon as practicable after donation in order to moreeffectively reduce or eliminate contaminating factors, including but notlimited to leucocytes and microaggregates.

The invention also involves a method for collecting and processing bonemarrow comprising harvesting bone marrow from a donor into a harvestingcontainer, such as a syringe or the like; transferring the harvestedbone marrow into a closed or sealable collection container; repeatingthe harvesting step and the transferring step until a sufficientquantity of bone marrow and/or stem cells are obtained, and collectingthe bone marrow in the collection container. The method may furtherinclude passing the harvested bone marrow in the first container througha first porous medium, the first porous medium comprising amicroaggregate filter having a pre-determined pore size or rating; andpassing the filtered bone marrow into a second closed or sealablecontainer.

In a preferred embodiment of the invention, transferring the harvestedbone marrow into a collection container may include passing the bonemarrow through a first fluid flow path into the collection container,closing the first flow path, opening a second fluid flow path between athird container containing anti-coagulant solution and the harvestingcontainer; passing anti-coagulant solution into the harvestingcontainer; expressing anti-coagulant solution from the harvestingcontainer, preferably into the third container; closing the second flowpath; and repeating the harvesting step and the transferring step untila sufficient quantity of bone marrow and/or stem cells are obtained.

In a most preferred embodiment of the invention, the transferring stepincludes repeating the harvesting step and the transferring step until asufficient quantity of bone marrow and/or stem cells are obtained, thenopening a fluid flow path between the first container and the thirdcontainer, and expressing the contents of the third container into thefirst container.

In a preferred embodiment of the invention, the bone marrow collectionand processing system is sealed and/or closed.

An exemplary bone marrow or biological fluid collection and processingsystem is shown in FIG. 1. The bone marrow processing system isgenerally denoted as 10. It may comprise a first container or collectionbag 11, said first container comprising at least one access port 12 andat least one discharge port 13; a second container (first satellite bag)14; and, interposed between the first container 11 and the secondcontainer 14, at least one filter assembly 15. Each of the assemblies orcontainers may be in fluid communication through tubing, preferablyflexible tubing, 20, 21, and 22. In accordance with the invention, thereis a first fluid flow path between the first container 11 and secondcontainer 14. A seal, valve, clamp 23, or transfer leg closure orcannula (not illustrated) may also be positioned in or on the tubing orin the collection and/or satellite bags. The seal (or seals) is openedwhen fluid is to be transferred between bags.

In a preferred embodiment of the invention, first container 11 alsoincludes at least one valve 24, preferably in fluid communication withaccess port 12. In the illustrated embodiment, valve 24 is in fluidcommunication with port 12 through conduit 22. Valve 24 permits accessto the collection bag; in a preferred embodiment of the invention, valve24 is or includes a connector adapted for communication with a syringeor the like, e.g., a Luer lock.

An apparatus or system according to the invention may also include oneor more manifolds 30. Manifold 30 may be variously configured to provideclosed and/or sealed fluid communication between a harvesting container(not shown) and first or collection container 11, to provide closedand/or sealed fluid communication between a harvesting container (notshown) and third container 34, to provide closed and/or sealed fluidcommunication between first container 11 and third container 34, toprovide fluid communication between first container 11 and a samplingcontainer (not shown), such as a syringe, or combinations thereof. Anembodiment of the invention is illustrated in FIG. 2.

In FIG. 2, manifold 30 is in fluid communication with first container 11through conduit 22. Manifold 30 includes port 31 configured to engagecomplementary structures on a harvesting container (e.g., a syringe, notshown). In a preferred embodiment of the invention, the harvestingcontainer includes a connector or the like suitable for engaging port31. Such connectors are well known in the art, and include but are notlimited to a Luer lock.

Manifold 30 may include a channel, conduit 32, or the like, that definesa fluid flow path between the harvesting container and the collectioncontainer. Manifold 30 may include a channel, conduit 33, or the like,that defines a fluid flow path between the harvesting container and athird container 34. Third container 34 preferably contains ananti-coagulant solution, such as heparin, CPDA-1, DMSO, or the like.Third container 34 may be directly attached to the manifold, may itselfinclude structures that function as a manifold as defined herein, or maybe connected to the manifold 30 through conduit 35. Manifold 30 mayinclude a channel, conduits 32 and 33, or the like, that defines a fluidflow path between the first container 11 and the third container 34.

In a system according to the invention, the flow path between the firstcontainer and the harvesting container is the second fluid flow path;between the harvesting container and the third container, the thirdfluid flow path; and between the first container and the thirdcontainer, the fourth fluid flow path.

In a preferred embodiment of the invention, the second, third and/orfourth fluid flow paths may include one or more valves, preferably influid communication with access port 31. The valves may be an elementintegral to a container or manifold, or may be connected to a containeror manifold. In the illustrated embodiment, valve 36 is interposed inthe fluid flow path between first container 11 and manifold 30, andvalve 37 is interposed in the fluid flow path between third container 34and manifold 30. In accordance with the invention, valves 36 and 37 maybe any valve, clamp, restrictor, diaphragm, connector, or the like thatselectively permits fluid flow therethrough, and is suitable for usewith a composition containing bone marrow and/or anti-coagulantsolution. In a preferred embodiment of the invention, valves 36 and 37are plunge valves, valves that open a channel when the plunger isdepressed. Suitable valves are commercially available under thetradename TRAC™ Valve.

As noted above, a system according to the invention also preferablyincludes one or more sampling ports. For example, ports 12, 13, and 31may function as sampling ports. In a preferred embodiment of theinvention, first container 11 includes sampling port 40. In a mostpreferred embodiment of the invention, the sampling port maintains theassembly or system in a closed and/or sealed condition. For example,sampling port 40 may include a re-sealable septum or diaphragm or thelike that permits the insertion of a needle into the first container,and re-seals the first container when the needle is withdrawn.

One skilled in the art will recognize that multiple harvestingcontainers and/or multiple manifolds may be incorporated into a systemaccording to the invention.

In accordance with the present invention, any number and combinations ofassemblies, porous media, containers, conduits, and methods of operationor processing are suitable. The invention comprises structures andmethods for harvesting a composition comprising bone marrow,transferring the harvested bone marrow to a collection container, andprocessing the collected bone marrow, more preferably into a compositioncontaining bone marrow suitable for use as a transplant graft. Oneskilled in the art will recognize that the invention as described heremay be reconfigured into different combinations, elements, and processeswhich are included within the scope of the invention.

A method according to the present invention includes harvesting acomposition comprising bone marrow, passing the bone marrow through asystem according to the present invention, and processing the bonemarrow into a composition suitable for a pre-determined end use, e.g.,suitable as a transplant graft, as a source of stem cells, or as acomposition suitable for use as part of research and development relatedto one or more constituents in the bone marrow composition. As will bereadily apparent to one skilled in the art, methods, steps, or aspecific sequence of steps according to the invention will be dictatedin part by the specific elements incorporated in a specific system. Forexample, methods relating to a system lacking a third container wouldnot include steps that involve the third container.

The following provides exemplary methods and specific exemplary steps toillustrate the function of an apparatus and system according to thepresent invention.

Once a donor is prepared for a bone marrow harvest procedure, bonemarrow from the donor may be drawn into a harvesting container, such asa syringe. Typically, about 5 cc to about 30 cc of bone marrow aredrawn. The harvesting container is then disconnected from the donor andattached to an apparatus according to the present invention. Forexample, a harvesting syringe may be connected to access port 12 onfirst or collection container 11 or on access port 31 on manifold 30.Collection container 11 typically includes a pre-determined amount ofanti-coagulant solution, and the harvested bone marrow is mixed with theanti-coagulant solution. In use, a biological fluid, e.g., bone marrow,is fed under sufficient pressure into the inlet of container 11 from anysuitable source of the biological fluid. For example, bone marrow may beharvested from a donor using a syringe or the like, and the bone marrowmay be injected from a syringe into the inlet of the collectioncontainer 11.

In the embodiments of the invention that include a manifold 30, theharvested bone marrow may be expressed or passed from the harvestingcontainer through port 31, through open valve 36, through conduit 22,and into collection bag 11. Valve 36 is then closed, valve 37 is thenopened, and anti-coagulant solution in third container 34 may beexpressed or drawn into the harvesting container through conduit 35,valve 37 conduit 33, and port 31. Once the harvesting container isflushed or rinsed with anti-coagulant solution, the solution may bereturned to third container 34, and the harvesting container is readyfor re-use, i.e., for drawing another 5–30 cc of bone marrow from thepatient.

The harvesting step and the transfer of the bone marrow composition tothe collection container may be repeated as often as desired, typicallyuntil up to about 1.5 liters or more of bone marrow is harvested.

Once the desired amount of bone marrow is harvested, if the systemincludes a third container 34, the contents of third container 34(typically including a mixture of anti-coagulant solution and bonemarrow rinsed from the harvesting container), may be expressed or passedinto first container 11, where it is mixed with the bone marrowcomposition collected in first container 11. In the embodiment of theinvention shown in FIGS. 1 and 2, a fluid flow path is opened betweenthird container 34 and first container 11, typically by opening valve37, closing port 31, opening valve 36, and opening port 12. Thecomposition in first container 11 at this point in the process isreferred to as the collected bone marrow composition.

At this point in the method, or earlier, it may be desirable to test orsample the composition in the first container 11. In this embodiment ofthe invention, a sampling device (not illustrated) may be insertedthrough re-sealable sampling port 40 and into the composition containedtherein. Once a sample is withdrawn from the container, sampling port 40preferably closes or re-seals.

The collected bone marrow may then be processed by opening orestablishing fluid communication between the first container 11 and thesecond container 14. By creating a pressure differential between firstcontainer 11 and second container 14, biological fluid will flow fromthe first container to the second container. In accordance with someembodiments of the invention, the bone marrow will be filtered asdesired if a filter assembly is interposed in the fluid flow pathbetween the first container 11 and the second container 14.

The invention also involves a method for harvesting, collecting, andprocessing bone marrow comprising harvesting bone marrow in a container,collecting bone marrow in a collection container; and passing the bonemarrow through a filter assembly and into a second container.

In general, donated bone marrow is processed as soon as practicable inorder to more effectively reduce or eliminate contaminating factors,including but not limited to leucocytes and microaggregates.

Each of the components of the assembly will now be described in moredetail below.

The containers which are used in the biological fluid processingassembly may be constructed of any material compatible with a biologicalfluid, such as bone marrow or a bone marrow component, and is capable ofwithstanding a sterilization environment. A wide variety of thesecontainers are already known in the art. For example, bone marrowcollection and satellite bags are typically made from plasticizedpolyvinyl chloride, e.g. PVC plasticized with dioctylphthalate,diethylhexylphthalate, or trioctyltrimellitate. The bags may also beformed from polyolefin, polyurethane, polyester, and polycarbonate.

As used herein, the tubing may be any conduit or means which providesfluid communication between the containers, and is typically made fromthe same flexible material as is used for the containers, preferablyplasticized PVC. The tubing may extend into the interior of thecontainer, and may be used as a siphon, for example. There may be anumber of tubes providing fluid communication to any individualcontainer, and the tubes may be oriented in a number of ways. Forexample, there may be at least two tubes oriented at the top of thecollection bag, or at the bottom of the bag, or a tube at each end ofthe bag.

Additionally, the tubes, assemblies, porous media, and containers, maybe oriented to define different flow paths. For example, when bonemarrow is processed, a first portion may flow along a first flow path,e.g., through a 850 micron filter and into a satellite bag (e.g., asecond container). Similarly, a second portion may flow along a secondflow path, e.g., through a 200 micron filter assembly, and into asatellite bag (e.g., a third container). Since independent flow pathsmay be present, biological fluids may flow concurrently, orsequentially.

A seal, valve, clamp, transfer leg closure, or the like is typicallylocated in or on the tubing. It is intended that the present inventionis not limited by the type of material used to construct the containersor the conduit which connects the containers.

As noted above, the bone marrow is passed through one or more porousmedia adapted to remove one or more predetermined constituents in thebone marrow. As used herein, adapted to remove refers to a physical,biological, chemical, or other arrangement or surface enhancementintended to effect the removal of the component. For example, it may bedesirable to remove all components in the bone marrow having a sizegreater than about 850 microns. In this embodiment, the porous mediawould be adapted to remove particles greater than about 850 microns byhaving a pore rating of about 850 microns. In a second example, it maybe desirable to remove leukocytes from the bone marrow. In thisembodiment, the porous media might be adapted to remove leukocytes bycoating the surface of a fibrous porous media with one or more elementsthat attract and/or bind leukocytes. In a third example, it may bedesirable to specifically remove stem cells from the bone marrow. Inthis embodiment, the porous media might be adapted to remove stem cellsby coating the surface of a fibrous porous medium with an antibody orthe like that specifically binds stem cells. It is intended that thepresent invention should be limited to a specific configuration of theporous medium.

The porous medium may be formed from any natural or synthetic fiber (orfrom other materials of similar surface area and pore size) compatiblewith blood. The porous medium may remain untreated. Preferably, thecritical wetting surface tension (CWST) of the porous medium is within acertain range as dictated by its intended use and/or by the type ofbiological fluid passing through the porous medium. The pore surfaces ofthe medium may be modified or treated in order to achieve a desiredproperty. Although the filter medium may remain untreated, the fibers ormembrane are preferably treated to make them even more effective forseparating one component from other components of bone marrow. Theporous medium is preferably treated in order to reduce or eliminateplatelet adherence to the medium. Any treatment which reduces oreliminates platelet adhesion is included within the scope of the presentinvention. Furthermore, the medium may be surface modified as disclosedin U.S. Pat. No. 4,880,548, incorporated herein by reference, in orderto increase the critical wetting surface tension (CWST) of the mediumand to be less adherent of platelets.

In the embodiments of the invention in which the porous medium includesfibers, the surface characteristics of the fiber may remain unmodified,or can be modified by a number of methods, for example, by chemicalreaction including wet or dry oxidation; by coating the surface bydepositing a polymer thereon; or by grafting reactions wherein thesubstrate or fiber surface is activated prior to or during wetting ofthe fiber surface by a monomer solution by exposure to an energy sourcesuch as heat, a Van der Graff generator, ultraviolet light, or tovarious other forms of radiation; or by subjecting the fibers to gasplasma treatment. A preferred method is a grafting reaction usinggamma-radiation, for example, from a cobalt source.

An exemplary radiation grafting technique employs at least one of avariety of monomers each comprising an ethylene or acrylic moiety and asecond group, which is preferred to be a hydrophilic group (e.g., —COOH,or —OH). Grafting of the fibrous medium may also be accomplished bycompounds containing an ethylenically unsaturated group, such as anacrylic moiety, combined with a hydroxyl group, preferably monomers suchas hydroxyethyl methacrylate (HEMA), or acrylic acid. The compoundscontaining an ethylenically unsaturated group may be combined with asecond monomer such as methyl acrylate (MA), methyl methacrylate (MMA),or methacrylic acid (MAA). Analogues with similar functionalcharacteristics may also be used to modify the surface characteristicsof fibers.

If desired, the flow rate of biological fluid through the filter can beregulated to obtain a total flow period of about 10 to 40 minutes byselecting the appropriate element diameter, element thickness, fiberdiameter, and density, and/or by varying the diameter of the tube eitherupstream or downstream of the filter, or both up and downstream.

All of these parameters can be varied; for example, the diameter of theporous medium could be reduced and the thickness increased whileretaining the same total quantity of fiber, or the fibers could belarger in diameter while increasing the total quantity of fiber, or thefibers could be packed as opposed to preformed into a cylindrical disc.Such variations fall within the purview of this invention.

As noted above, as the bone marrow is expressed from the collection bag,it may be processed through a device having a leucocyte depletionelement in order to reduce the leucocyte content of the bone marrow. Inaccordance with the invention, the porous medium for removing leucocytesfrom the bone marrow may comprise a leucocyte removal element or porousmedium having an average diameter of from about 1 to about 4 μm,preferably from about 2 to about 3 μm. Polybutylene terephthalate (PBT)web, which is a preferred material, may be hot compressed to a voidsvolume of about 65% to about 90% and preferably to about 73% to about88.5%.

The biological fluid may be supplied in any suitable quantity consistentwith the capacity of the overall device and by any suitable means, e.g.,in a batch operation by, for example, a collection bag connected to anexpresser or a syringe, or in a continuous operation as part of, forexample, an apheresis system. A source of biological fluid may alsoinclude an apheresis system, and/or may include a system in whichbiological fluid is re-circulated through the system.

Further, a bone marrow system according to the invention may include anaccess port 12 configured as or in fluid communication with a manifoldvalve, said manifold valve having one or more ports adapted forcommunicating with a syringe. In this embodiment of the invention, bonemarrow may be harvested by multiple technicians using multiple syringes,and the bone marrow in multiple syringes may be transferred to thecollection bag simultaneously or consecutively.

The present inventive device may similarly be part of an apheresissystem. The biological fluid to be processed may be handled in either abatch or continuous manner. The sizes, nature, and configuration of thepresent inventive device can be adjusted to vary the capacity of thedevice to suit its intended environment.

Under certain circumstances, it may be desirable to maximize therecovery of a biological fluid retained or entrapped in various elementsof the biological fluid processing system. For example, under typicalconditions, using a typical device, the biological fluid will drainthrough the system until the flow is stopped, leaving some of the fluidin the system. In one embodiment of the invention, the retained fluidmay be recovered by using at least one gas inlet and/or at least one gasoutlet.

The invention may also comprise at least one gas inlet, and/or at leastone gas outlet. For example, first container 11 may also be in fluidcommunication with a gas inlet, a microaggregate filter assembly 15, anda gas outlet. The assembly may also include additional containers, flowpaths, and porous media. The gas outlet is a porous medium which allowsgas that may be present in a biological fluid processing system when thebiological fluid is processed in the system, out of the system. The gasinlet is a porous medium which allows gas into a biological fluidprocessing system.

As used herein, gas refers to any gaseous fluid, such as air, sterilizedair, oxygen, carbon dioxide, and the like; it is intended that theinvention is not to be limited to the type of gas used.

The gas inlet and gas outlet are chosen so that the sterility of thesystem is not compromised. The gas inlet and the gas outlet areparticularly suited for use in closed systems, or may be used later, forexample, within about 24 hours of a system being opened.

The gas inlet and the gas outlet each comprise at least one porousmedium designed to allow gas to pass therethrough. A variety ofmaterials may be used, provided the requisite properties of theparticular porous medium are achieved. These include the necessarystrength to handle the differential pressures encountered in use and theability to provide the desired filtration capability while providing thedesired permeability without the application of excessive pressure. In asterile system, the porous medium should also preferably have a porerating of about 0.2 micrometer or less to preclude bacteria passage.

The gas inlet and gas outlet may comprise a porous medium, for example,a porous fibrous medium, such as a depth filter, or a porous membrane orsheet. Multilayered porous media may be used, for example, amultilayered microporous membrane with one layer being liquophobic andthe other liquophilic. Particularly preferred are skinless,substantially alcohol-insoluble, hydrophilic polyamide membranes, suchas those described in U.S. Pat. No. 4,340,479.

The rate of air flow through the gas outlet or the gas inlet can betailored to the specific biological fluid or fluids of interest. Therate of air flow varies directly with the area of the porous medium andthe applied pressure. Generally, the area of the porous medium isdesigned to enable the biological fluid processing system to be primedin a required time under the conditions of use. For example, in medicalapplications it is desirable to be able to prime an intravenous set infrom about 30 to about 60 seconds. In such applications as well as inother medical applications, the typical porous medium is a membrane,which may be in the form of a disc which has a diameter from about 1 mmto about 100 mm, preferably from about 2 mm to about 80 mm, and morepreferably from about 3 mm to about 25 mm.

In accordance with the invention, the processing system may be providedwith a gas inlet to permit the introduction of gas into the system,and/or with a gas outlet to permit gases in the various elements of thesystem to be separated from the biological fluid to be processed. Thegas inlet and the gas outlet may be used together in connection with atleast one assembly, porous medium, or container in the system, or theymay be used separately.

To that end, a gas inlet or gas outlet may be included in any of thevarious elements of the biological fluid processing system. By way ofillustration, a gas inlet or gas outlet may be included in at least oneof the conduits which connect the different containers, in a wall of thecontainers that receive the processed biological fluid, or in a port onor in one of those containers. The gas inlet or gas outlet may also beincluded on or in a combination of the elements mentioned above. Also,an assembly or porous medium may include one or more gas inlet or gasoutlet as described above. Generally, however, it is preferred toinclude a gas inlet or gas outlet in the conduits which connect thecontainers or in the functional medical device. Included within thescope of the invention is the use of more than one gas inlet or gasoutlet in any conduit, receiving container, assembly, or porous medium.

It will be apparent to one skilled in the art that the placement of agas inlet or a gas outlet may be optimized to achieve a desired result.For example, it may be desirable to locate the gas inlet upstream of aporous medium and in or as close to the first container as is practicalin order to maximize the recovery of biological fluid. Also, it may bedesirable to locate the gas outlet downstream of the porous medium andas close to the receiving container as is possible in order to maximizethe volume of gas that is removed from the system.

Such placement of the gas inlet or gas outlet is particularly desirablewhere there is only one gas inlet or gas outlet in the system.

In accordance with the invention, recovery from the various elements ofthe biological fluid processing system may be maximized. For example,bone marrow is subjected to a processing step, resulting in filteredbone marrow. Blood product that has become entrapped in these elementsduring processing may be recovered either by passing purge gas throughthe conduits and porous media, or by creating at least a partial vacuumin the system to draw out the retained blood product and to permit it todrain into the appropriate receiving container or assembly.

The purge gas may be from any of a number of sources. For example, thebiological fluid processing system may be provided with a storagecontainer for the storage of the purge gas, the purge gas may be the gasthat was removed from the system during the processing function, or thepurge gas may be injected aseptically into the system from an outsidesource (e.g., through a syringe). For example, it may be desirable touse sterile purge gas that has been sterilized in a separate containerapart from the biological fluid processing system.

In accordance with the invention, a clamp, closure, or the like may bepositioned on or in any or all of the conduits in order to facilitate adesired function, i.e., establishing a desired flow path for biologicalfluid or gas.

The gases separated by the gas outlet may be vented from the system, orthey may be collected in a gas container (not shown) and returned to thesystem as a purge gas to facilitate the recovery of biological fluidthat becomes trapped in the various components of the system.

After the system is primed and the gas outlet is inactivated, the clampadjacent to the containers or assembly is opened to allow the containersto fill with processed biological fluid. This continues until thecollection bag 11 collapses. In order to recover the very valuablebiological fluid retained in the system, ambient air or a sterile gasmay enter the system through gas inlet. If gas inlet is a manual inletmeans, a closure is opened or a clamp released; if the gas inlet isautomatic, the pressure differential between the gas inlet and thecontainers will cause the air or gas to flow through the conduits,through the porous media, and towards the respective containers. In theprocess, retained biological fluid that is trapped in those elementsduring processing are recovered from those components and collected inthe containers. It should be noted that the purge air or gas ispreferably separated from the biological fluid at gas outlet so thatlittle, if any, purge gas will be received by the containers. This maybe accomplished by clamping the conduit downstream of the gas outlet. Inanother embodiment of the invention, the purge air or gas may beseparated from the system through a gas outlet located in the bagitself.

A system according to the present invention may be used in conjunctionwith other assemblies or porous media, including filtration and/orseparation devices, e.g., a device for removing leucocytes from aplatelet-containing solution or concentrate.

A number of additional containers may be in communication with thebiological fluid processing system, and can be utilized to definedifferent flow paths. For example, an additional satellite bagcontaining physiological solution may be placed in communication withthe biological fluid processing system upstream of the filter assembly,and the solution may be passed through the filter assembly so that thebiological fluid that was held up in the assembly can be collected.

It will be appreciated that when the biological fluid from thecollection bag 11 is expressed towards the containers, some of thebiological fluid may be trapped in the conduits and/or the porousmediums. For example, 8 cc to 35 cc is typically retained in the system;but as little as 2 cc to as much as 150 cc or more may be retained insome types of systems.

In an embodiment of the invention (not shown), air or gas may be storedin at least one gas container; upon opening of valve or clamp means inthe conduits, gas can be fed through them to purge the conduits andassemblies, thereby facilitating the recovery of biological fluid thatmay have been trapped during processing.

Preferably, the purge air or gas is fed to the conduits at a point asclose as is reasonably possible to container 11 to maximize the volumeof biological fluid recovered. The air or gas container is preferablyflexible so that the gas therein may be fed to the system merely bysimple compression. The biological fluid containers and the air or gascontainers may be composed of the same material.

In an embodiment of the invention (not shown), priming fluid may bestored in at least one container; upon opening of valve or clamp meansin the conduits, priming fluid can be fed through them to prime and/orwet the conduits and assemblies, thereby facilitating the function andoperation of the various elements in the system. Priming fluid includesbut is not limited to a solution or composition containing bone marrow,an anti-coagulant solution, or any solution compatible with bone marrowand effective as a wetting or priming solution.

It will be apparent to one skilled in the art that the placement andconnection of a source of priming fluid to the system may be optimizedto achieve a desired result.

Priming, as used herein, refers to wetting or priming the inner surfacesof a device or assembly prior to its actual use allowing a separateassembly to be injected into the system. A valve or clamp is opened toallow fluid to flow through the assembly; then, with the passage offluid through the assembly, gas downstream of the fluid is expelledthrough the gas outlet until fluid reaches a branching element, at whichpoint the clamp is closed. With the clamp in a closed position, theconnector downstream of the gas outlet may be opened or readied for usewithout fluid in the assembly dripping through the connector.

In accordance with the invention, the biological fluid collection andprocessing assembly should be able to withstand rigorous sterilization,typically consisting of radiation sterilization (at about 2.5 megarads),and/or autoclaving (at about 110° C. to about 120° C. for about 15 to 60minutes).

There may be a clamp or the like between the collection bag 11 and theflexible tubing or within the tubing, to prevent the flow of the bonemarrow from entering the wrong conduit.

Movement of the biological fluid through the system is effected bymaintaining a pressure differential between the source of the biologicalfluid and the destination of the biological fluid (e.g., a containersuch as a satellite bag or a needle on the end of a conduit). The systemof the invention is suitable for use with conventional devices forestablishing the pressure differential, e.g., an expresser. Exemplarymeans of establishing this pressure differential may be by gravity head,applying pressure to the collection bag (e.g., by hand or with apressure cuff), or by placing the other container (e.g., satellite bag)in a chamber (e.g., a vacuum chamber) which establishes a pressuredifferential between the collection bag and the other container. Alsoincluded within the scope of the invention may be expressors whichgenerate substantially equal pressure over the entire collection bag.

While the invention has been described in some detail by way ofillustration and example, it should be understood that the invention issusceptible to various modifications and alternative forms, and is notrestricted to the specific embodiments set forth in the Examples. Itshould be understood that these specific embodiments are not intended tolimit the invention but, on the contrary, the intention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention.

1. A method for collecting and processing bone marrow comprisingtransferring harvested bone marrow into a sealed first container bypassing the harvested bone marrow through at least one manifold;processing the harvested bone marrow; passing the processed bone marrowinto a second container; passing a solution housed in a third containerthrough the manifold; and then returning the solution to the thirdcontainer.
 2. The method of claim 1 further comprising passing thesolution in the third container into the first container.